Introdução:Uma das causas da doença cardiovascular do diabético é a dislipidemia associada ao diabetes mellitus tipo 2 (DM2), caracterizada basicamente por hipertrigliceridemia e baixa concentração de HDL-colesterol. O bom controle da glicemia geralmente resulta em diminuição dos triglicerídeos plasmáticos, mas há controvérsia na literatura quanto aos níveis de HDL-colesterol e outros parâmetros lipídicos. As transferências lipídicas para a HDL têm importante função na sua formação e remodelamento e no seu papel antiaterogênico do transporte reverso do colesterol. A transferência de lípides entre as lipoproteínas é bidirecional e depende da estrutura e concentração da lipoproteína doadora e receptora, assim como da ação das proteínas de transferências, CETP e PLTP. Objetivo:Investigar as relações dos níveis glicêmicos com as transferências lipídicas para a HDL e com outros parâmetros do metabolismo lipídico em pacientes com DM2. Métodos:143 pacientes com DM2, que não estavam usando drogas hipolipemiantes, foram selecionados e separados em dois grupos: grupo com hemoglobina glicada (HbA1c) <= 6,5% (n=62) e grupo com HbA1c > 6,5% (n=81). O método in vitro de transferência lipídica para a HDL foi realizado através da incubação sob agitação, por 1 hora, de uma nanoemulsão doadora contendo colesterol esterificado e não esterificado, fosfolipídios e triglicerídeos, marcados radioativamente, com o plasma do paciente, seguido de precipitação química e contagem dos lipídios radiomarcados transferidos para HDL. Outras determinações plasmáticas do metabolismo lipídico também foram realizadas. Foi também verificado o percentual de pacientes nos dois grupos com o perfil lipídico dentro das metas estabelecidas pela American Diabetes Association. Pacientes com ou sem uso de insulina e com níveis maiores e menores de ácidos graxos livres (AGL) foram comparados quanto aos parâmetros estudados. Resultados:O grupo HbA1c > 6,5% apresentou maior trigliceridemia (205±115 vs 140±54mg/dl; p < 0,0001) e colesterol não esterificado (36,4±7,6 vs 33,7±5,7mg/dl; p > 0,05), além de apresentar maior concentração de triglicerídeos na partícula de HDL (9,2±2,8 vs 8,1±2,3%; p < 0,05), do que o grupo HbA1c<=6,5%. As concentrações de triglicerídeos, colesterol total e não esterificado e também o colesterol da fração não-HDL correlacionaram-se positivamente com a HbA1c (r=0,25, r=0,19, r=0,18, r=0,17, respectivamente, p < 0,05). A concentração de colesterol esterificado da HDL correlacionou-se negativamente com a HbA1c (r=-0,19, p < 0,05). Os pacientes com HbA1c <= 6,5% atingiram mais a meta trigliceridêmica do que os com HbA1c > 6,5% (66 vs 37%; p < 0,001). Os pacientes com maiores níveis de AGL tiveram maior trigliceridemia (196±105 vs 153±81 mg/dl; p < 0,01) e atividade de LCAT (1,36±0,10 vs 1,29±0,10 470/390nm; p < 0,01), além de uma maior concentração de triglicerídeos na partícula de HDL (9,4±2,9 vs 7,8±2,0%; p < 0,001), em relação aos pacientes com menores níveis de AGL. A transferência dos quatro lipídios da nanoemulsão para a HDL foi igual na comparação de todos os grupos deste estudo. Conclusão:Os resultados deste estudo mostraram pela primeira vez que junto com os triglicerídeos, o colesterol não esterificado é também um marcador de pobre controle glicêmico em pacientes com DM2 / Introduction:One cause of cardiovascular disease (CVD) of patients with type 2 diabetes mellitus (T2DM) is diabetic dyslipidemia, characterized primarily by hypertriglyceridemia and low HDL-cholesterol. Good blood glucose control usually results in decreased plasma triglycerides, but there is controversy in the literature as to the levels of HDL-cholesterol and other lipid parameters. Lipid transfers to HDL play an important role in the formation and remodeling of HDL and on its antiatherogenic role of reverse cholesterol transport. The transfer of lipids between lipoproteins is bidirectional and depends on the structure and concentration of the donor and acceptor lipoprotein as well as the action of the transfers proteins, PLTP and CETP. Objective:Investigate the relationship of blood glucose levels with lipid transfers to HDL and other parameters of lipid metabolism in patients with T2DM. Methods:143 patients with T2DM, who were not using lipid-lowering drugs, were selected and divided into two groups: group with glycated hemoglobin (HbA1c) <= 6.5% (n=62) and group with HbA1c > 6.5% (n=81). In vitro lipid transfers to HDL was performed by 1 hour incubation under stirring of a donor nanoemulsion containing radioactively labeled unesterified and esterified cholesterol, phospholipids and triglycerides with whole patient plasma, followed by chemical precipitation and counting of radiolabeled lipids transferred to HDL. Other determinations of plasma lipid metabolism were also performed. It was also checked the percentage of patients in both groups with lipid profile within the targets set by the American Diabetes Association criteria. Patients with or without insulin use and with higher and lower free fatty acids (FFA) levels were also compared for the studied parameters. Results:HbA1c>6.5% group had higher triglyceridemia (205 ± 115 vs 140 ± 54 mg/dl, p<0.0001) and unesterified cholesterol (36.4 ± 7.6 vs 33.7 ± 5.7 mg/dl, p<0.05) than the HbA1c<=6.5% group. The concentrations of triglycerides, total and unesterified cholesterol and also non-HDL cholesterol was positively correlated with HbA1c (r=0.25, r=0.19, r=0.18, r=0.17, respectively, p<0,05). The concentration of esterified cholesterol from HDL was negatively correlated with HbA1c (r = -0.19, p<0.05). Patients with HbA1c<=6,5% achieved more the triglyceridemic target than the patients with HbA1c>6.5% (66 vs 37%, p<0.001). Patients with higher levels of FFA had higher triglycerides levels (196 ± 105 vs 153 ± 81 mg/dl, P<0.01) and LCAT activity (1.36 ± 0.10 vs 1.29 ± 0.10 470/390nm, p<0.01), in addition to a higher concentration of triglycerides in the HDL particle (9.4 ± 2.9 vs 7.8 ± 2.0%, p <0.001). The transfer of the four lipid of the nanoemulsion to HDL was equal in the comparison of all the studied groups. Conclusion:The results of this study showed for the first time that unesterified cholesterol, along with triglycerides, is also a marker of poor glycemic control in patients with T2DM

Estima-se que 3% da população mundial esteja infectada pelo vírus da hepatite C (HCV). O HCV tem como alvo o tecido hepático e a maioria dos pacientes infectados desenvolvem infecção crônica. Nos últimos anos, estudos in vitro têm demonstrado interações entre o miRNA-122 (miR-122) da célula hospedeira e dois sítios localizados na região 5\' UTR do genoma do vírus da hepatite C (HCV), os quais são essenciais ao processo de replicação viral. O miR-122 é altamente expresso no fígado, onde atua na regulação do metabolismo de lipídios juntamente com outro miRNA, o miRNA-33a (miR-33a), porém, o mecanismo envolvido nesta regulação ainda é pouco conhecido. Sabe-se que a infecção pelo HCV altera a expressão de genes envolvidos na biossíntese e transporte de lipídios, resultando na estimulação do metabolismo de lipídios e criando um ambiente favorável para sua replicação. Neste contexto os objetivos deste trabalho foram avaliar a expressão de miR-33a e miR-122 em indivíduos cronicamente infectados pelo HCV-1 e HCV-3 em amostras obtidas antes do início da terapia. Os miRNAs foram isolados a partir de amostras de sangue periférico e de tecido hepático. A quantificação da expressão relativa de ambos miRNAs foi pela técnica de PCR em tempo real. Os níveis de miR-33a no sangue periférico foram mais elevados do que no tecido hepático em indivíduos infectados pelo HCV-1(p < 0,0001) e HCV-3 (p=0,0025). Observou-se uma correlação inversa entre os níveis de miR-33a no sangue periférico e tecido hepático dos indivíduos infectados pelo HCV-1 (r=-0,281, p=0,039) e correlação positiva para os indivíduos infectados pelo HCV-3 (r=0,9286, p < 0,0001). Correlação inversa entre os níveis hepáticos de miR-33a com o nível sérico de insulina (r=-0,371, p =0,005) nos indivíduos infectados pelo HCV-1 e correlação positiva entre os níveis no sangue periférico com os níveis séricos de GGT (r=0,553, p=0,049) foram observadas. Em relação ao miR-122, de maneira geral o nível hepático foi mais elevado do que o sérico (p < 0,0001). Entretanto, o nível hepático de miR-122 em indivíduos infectados pelo HCV-3 foi maior quando comparado aos infectados pelo HCV-1 (6,22 vezes, p < 0,001). Uma correlação inversa entre os níveis séricos de ApoA-II e os níveis de expressão de miR-122 no sangue (r=-0,330; p=0,014) e tecido hepático (r=-0,311; p=0,020) foi observada nos pacientes infectados pelo HCV-1. Os pacientes infectados pelo HCV- 3 mostraram correlação positiva entre os níveis hepáticos de miR-122 e os níveis de HDL (r=0,412, p=0,036) e insulina (r=0,478, p=0,044). O miR-33a e o miR-122 atuam regulando genes que controlam o metabolismo dos lipídios no fígado. Até o presente momento, não existem relatos que associem a expressão do miR-33a e do miR-122 com o perfil lipídico na infecção pelo HCV. Além disso, o acúmulo de lipídio (esteatose) intensamente descrito na infecção pelo HCV-3 pode sugerir interação diferenciada desse genótipo com os mecanismos envolvidos na regulação do metabolismo lipídico, envolvendo o miR-33a e miR-122 / The prevalence of infection by hepatitis C virus (HCV) is about 3% of the world population. HCV targets the liver tissue and the majority of infected patients develop chronic infection. In recent years, in vitro studies have demonstrated interactions between miRNA-122 (miR-122) the host cell to two places located in the 5\' untranslated region of the HCV genome which are essential for virus replication process. miR-122 is highly expressed in the liver, which has been implicated as a fatty acid metabolism regulator. Another mine has also been described as a key regulator of lipid metabolism, miRNA-33a (miR-33a), however, the mechanisms involved in this regulation are still little known. It is known that HCV infection changes the expression of genes involved in the biosynthesis and transport of lipids, resulting in stimulation of the lipid metabolism and creating a favorable environment for replication of the virus. To our knowledge, there are no reports linking the expression of miR-33a with lipid profile in HCV infection. In this context the objectives of this study were to evaluate the expression of miR-33a and miR-122 in chronically infected individuals with HCV-1 and HCV-3 in samples obtained prior to initiation of therapy. MiRNAs were isolated from peripheral blood samples and liver tissue. The quantification of relative expression of both miRNAs was by PCR in real time. MiR-33a levels in peripheral blood were higher than in liver tissue in patients infected with HCV-1 (p < 0.0001) and HCV-3 (p=0.0025). Levels in the peripheral blood of miR-33a were lower in patients infected with HCV-3 (p=0.0169). There was an inverse correlation between hepatic levels of miR-33a with serum insulin levels (p=0.005) in individuals infected with HCV-1 and a positive correlation between the levels in the peripheral blood serum levels of GGT (p=0.049). Hepatic levels of miR-122 were higher than the levels in the peripheral blood of individuals infected by HCV-1 and HCV-3 (p < 0.0001). Hepatic miR-122 levels were higher in patients infected with HCV-3 than those infected with HCV-1 (6.22 times, p < 0.001). There was a positive correlation between miR-122 levels in the blood and liver tissue of patients infected with HCV-1 (r=0.302, p=0.026). An inverse correlation between serum ApoA-II was observed in these patients the levels of expression of miR-122 in blood (r=-0.330; p =0.014) and liver tissue (r=-0.311; p=0.020). Patients infected with HCV-3 showed a positive correlation between hepatic miR-122 levels to HDL levels (r=0.412, p=0.036) and insulin levels (r=0.478, p=0.044). The miR-33a and miR-122 act by regulating genes that control lipid metabolism in the liver. The different interactions with lipid metabolism exerted by HCV-3 may explain why his relationship with the miR-33a and miR-122 was different when compared with HCV-1

A Arginina (Arg) regula a secreção de GH e insulina, e é o único precursor biológico do NO. Previamente demonstramos que animais tratados cronicamente com Arg (35mg/dia) desenvolvem resistência à insulina (RI), e o presente estudo investigou as suas bases moleculares. A RI baseou-se na redução da atividade e/ou expressão do IRS 1/2 e Akt, e do conteúdo de GLUT4; sendo o GH crucial na gênese desses efeitos. Doses mais elevadas de Arg (70mg/dia/30 dias), a maior geração de NO e a melhora do fluxo sangüíneo reverteram este quadro. Experimentos com células musculares demonstraram que a Arg estimula o metabolismo de glicose e lipídios, via NO/c-GMP. Esses achados indicam que a Arg pode ser benéfica para o tratamento de distúrbios metabólicos, como obesidade e DM2; e que ao estimular a secreção de GH, em doses adequadas, seria eficaz na terapia de distúrbios da secreção deste hormônio. Todavia, estudos adicionais são necessários para investigar a melhor dose e os efeitos crônicos in vivo da Arg, uma vez que o GH em excesso apresenta um efeito diabetogênico importante. / Arginine (Arg) regulates the secretion of GH and insulin, and it is the main biological precursor of NO. We have previously shown that animals chronically-treated with Arg (35 mg/day) developed insulin resistance (IR), and this study investigated its molecular basis. The RI relies on the reduction of the activity and/or expression of IRS 1/2 and Akt, and of the GLUT4 content; and GH has a crucial role in the genesis of these effects. Higher doses of Arg (70 mg/dia/30 days), the increased NO generation and the improvement of the blood flow reversed the RI. Experiments with muscle cells showed that Arg stimulates glucose and lipids metabolism, via NO/c-GMP activation. These findings indicate that Arg may be beneficial for the treatment of metabolic disorders, such as obesity and T2DM, and by stimulating GH secretion, Arg can, in appropriate doses, be effective for the therapy of GH secretion disorders. However, further studies are needed to investigate the best dose and the chronic effects of Arg in vivo, since that GH in excess is potentially diabetogenic.

Orientador: Dora Maria Grassi Kassisse / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T06:22:42Z (GMT). No. of bitstreams: 1 Ishizu_LarissaYuri_M.pdf: 1236160 bytes, checksum: d3b83c5f6882f0bbaa5b2be15aa753a0 (MD5) Previous issue date: 2012 / Resumo: Dados do Laboratório de Estudo do Estresse revelaram que a produção de lactato por adipócitos isolados de tecido adiposo visceral de humanos eutróficos e obesos mórbidos está sob estímulo de adrenoceptores ?1. Observou-se também hiperlactatemia no jejum de obesos mórbidos, aumento na liberação de lactato e na lipólise basal e estimulada em adipócitos viscerais isolados destes indivíduos, comparados com eutróficos. Porém dados de nosso laboratório e da literatura sugerem uma relação antagônica entre lipólise e lactatogênese, da qual participa o receptor GPR81, o qual está envolvido com lactato, inibindo a lipólise. Dados da literatura também sugerem a supressão da lipogênese e maior lipólise basal e estimulada dos adipócitos de obesos, com aumento da expressão da aquaporina 7, pelo qual o glicerol gerado na lipólise, é liberado. Por outro lado, estudos da literatura relatam a ocorrência da reesterificação dos produtos liberados após a lipólise de triacilgliceróis (TAGs) no tecido adiposo, na obesidade. Possivelmente, este seria o processo que promoveria a manutenção de grandes estoques de TAGs no tecido adiposo, apesar da maior lipólise e menor lipogênese. Frente aos nossos dados funcionais e os presentes na literatura sobre tecido adiposo visceral na obesidade, o objetivo deste estudo foi detectar alterações no metabolismo glicídico e lipídico neste tecido isolado de humanos obesos, em comparação com eutróficos, sob o enfoque da expressão gênica. Para tanto, quantificamos a expressão de genes envolvidos com a lactatogênese (lactato desidrogenase A, LDHA), lipogênese (acetil-CoA carboxilase, ACACA; glicerol quinase, GK; lipoproteína lipase, LPL) e lipólise (lipase hormônio sensível, LIPE; fosfodiesterase 3b, PDE3b; aquaporina 7, AQP7), e o gene relacionado com a inibição da lipólise via lactato (receptor-órfão acoplado à proteína G 81, GPR81) através do Real- Time PCR. Os resultados obtidos mostraram que o tecido adiposo de mulheres obesas expressa significativamente 49% a mais o gene LIPE e 66% a mais o gene LPL o de que mulheres eutróficas (p<0,05), enquanto que no tecido adiposo de homens não foi encontrada diferença significativa, apenas uma tendência a aumento do gene LPL nos obesos, comparados com eutróficos. Os adipócitos isolados do tecido adiposo visceral de homens obesos são morfometricamente maiores que os provenientes de eutróficos tendo como provável fator o aumento da expressão de LPL sem ser acompanhado de alterações na expressão de LIPE. Por outro lado, os adipócitos isolados do tecido adiposo visceral de mulheres obesas não apresentaram alterações morfométricas quando comparados aos adipócitos isolados de eutróficas, estes resultados são explicados pela análise da expressão dos genes LPL e LIPE do tecido adiposo desta região que se apresentou significativamente elevada em obesas. Desta forma, para este tecido estudado, existem alterações, dependente de gênero, que devem ser consideradas para estudos futuros sobre a obesidade. Neste trabalho, com as condições e a população estudada, referente a obesos e eutróficos de ambos os gêneros, podemos indicar as seguintes conclusões: a expressão de enzimas relacionadas à lipólise e a lipogênese em adipócitos isolados da região visceral de obesos é dependente do gênero enquanto que não há alterações significativas na expressão gênica relacionada à lactatogênese / Abstract: Previous data from the Laboratory of Stress Study showed that lactate production by adipocytes isolated from visceral adipose tissue of human normal and morbidly obese is under ?1-adrenoceptor stimulation. It was also observed hyperlactatemia in fasting from morbidly obese, an increase basal and stimulated lactate and glycerol production in visceral adipocytes isolated from these individuals, compared with normal weight. But data from our laboratory and the literature suggest an antagonistic relationship between lipolysis and lactatogênese, which participates in the GPR81 receptor, which is involved with lipolysis inhibition by lactate. Literature data also suggest the suppression of lipogenesis and increased basal and stimulated lipolysis in adipocytes of obese, with increased expression of aquaporin 7, whereby the glycerol generated in lipolysis, is released. Furthermore, published studies have reported the occurrence of re-esterification of the products released after lipolysis triacylglycerols (TAGs) in adipose tissue, in obesity. Possibly, this would be the process that would promote the maintenance of large stocks of TAGs in adipose tissue, despite the increased lipolysis and reduced lipogenesis. Front of our functional data and the literature on visceral adipose tissue in obesity, the aim of this study was to detect changes in glucose and lipid metabolism in this tissue isolated from obese humans, compared with normal weight, with a focus on gene expression. To this end, we quantified the expression of genes involved in lactatogênese (lactate dehydrogenase A LDHA), lipogenesis (acetyl- CoA carboxylase, ACACA, glycerol kinase, GK, lipoprotein lipase, LPL) and lipolysis (hormone sensitive lipase, LIPE; phosphodiesterase 3b , PDE3b; aquaporin 7 AQP7), and the gene related to the inhibition of lipolysis via lactate (orphan receptor-G protein coupled 81, GPR81) using Real-Time PCR. The results showed that adipose tissue isolated from obese women expressed significantly 49% more gene LIPE and 66% more LPL gene that women with normal weight (p <0.05), whereas in men no significant difference was found, only a tendency towards increased LPL gene in obese compared with normal weight. The isolated adipocytes visceral adipose tissue of obese men morphometrically are larger than those from normal weight bearing as the most probable cause increased expression of LPL without being accompanied by alterations in the expression of LIPE. Moreover, the isolated adipocytes visceral adipose tissue of obese women showed no morphological changes compared to normal weight of isolated adipocytes. These results are explained by analysis of gene expression of LPL and LIPE adipose tissue in this region which was significantly higher in obese women. Thus, there are changes, in this tissue studied, dependent on gender, which should be considered for future studies on obesity. In this work, the conditions and the population studied, referring to obese and normal for both genders, we can state the following conclusions: the expression of enzymes related to lipolysis and lipogenesis in adipocytes isolated from visceral fat of obese people is dependent on the gender while not there are significant changes in enzyme expression related to lactatogenesis / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Made available in DSpace on 2014-08-20T14:37:49Z (GMT). No. of bitstreams: 1 dissertacao_simone_halfen.pdf: 955737 bytes, checksum: 7d2e65aa98a74306ae8fbeea421563a7 (MD5) Previous issue date: 2012-02-27 / The essential fatty acids alpha-linolenic acid (α-LNA 18:3 n-3) and linoleic acid (LA 18:2 n-6) fatty acids are precursors of polyunsaturated very long chain (PUFA-VLC) families omega-3 and omega-6. These fatty acids when consumed during pregnancy are important for brain development and adaptation of the retina and fetal tissues, influencing the metabolic pathways of fatty acids in the offspring. Based on this, we seek to verify the relationship of the influence of the ratio of omega 6/3 in the diet of pregnants with the metabolic profile of fatty acids and liver tissue in subsequent generations. For this we used 48 rats (Rattus norvegicus) Wistar, 36 females and 18 males for the founder generation (G0) with the control group (CONT) received a diet with fat source as soybean oil and omega group (OM) received the diet with linseed oil. From these groups, the animals were selected for the training of two generations (F1 and F2). The animals receiving the CONT followed the control diet while the OM group was divided into two new groups, one receiving control diet (OM/CONT) and the other keeping the feed omega (OM/OM). The incorporation of fatty acids (FA) of omega-3 was higher in liver of rats in the group OM, while the FA of omega-6 had a greater uptake of the liver tissue of the CONT group. A major change can be observed in the amount of oleic AG in liver tissue, since the merger occurred higher in the group CONT and the lowest in OM/CONT, demonstrating that the diet provided to the G0 may have somehow influenced the incorporation of generations F1 and F2. This remodeling enzyme was also sufficient to change the outcome of NEFA, which demonstrated decreased lower lipid mobilization in the same group. / Os ácidos graxos essenciais alfa-linolênico (α-LNA 18:3n-3) e linoléico (LA 18:2n-6) são precursores de ácidos graxos poli-insaturados de cadeia muito longa (AGPI-CML) das famílias ômega-3 e ômega-6. Estes ácidos graxos quando consumidos durante a gestação são importantes para o desenvolvimento do cérebro e retina e na adaptação de tecidos fetais, influenciando as vias metabólicas dos ácidos graxos na prole. Baseado nisso, buscou-se verificar a relação da influência da proporção ômega 6/3 na dieta da gestante com o perfil metabólico e de ácidos graxos do tecido hepático nas gerações subsequentes. Para isso foram utilizados 48 ratos (Rattus norvegicus) da cepa Wistar/UFPel, sendo 36 fêmeas e 12 machos para a geração fundador (G0), com o grupo controle (CONT) recebendo uma dieta tendo como fonte lipídica o óleo de soja e o grupo ômega (ÔM) que recebeu a dieta com óleo de linhaça. A partir destes grupos, os animais foram selecionados para a formação de mais duas gerações (F1 e F2). Os animais do CONT seguiram recebendo a dieta controle enquanto o grupo ÔM foi dividido em dois novos grupos, um recebendo dieta controle (ÔM/CONT) e o outro mantendo a dieta ômega (ÔM/ÔM). A incorporação de ácidos graxos (AGs) da família ômega-3 foi superior no fígado das ratas do grupo ÔM, enquanto os AGs da família ômega-6 tiveram uma maior incorporação do tecido hepático do grupo CONT. Uma mudança importante pode ser observada na quantidade do AG oléico no tecido hepático, pois a incorporação maior aconteceu no grupo CONT e a menor no ÔM/CONT, demonstrando que a dieta fornecida para a G0 pode ter influenciado de alguma forma a incorporação das gerações F1 e F2. Esta remodelação enzimática foi suficiente também para mudar o resultado do NEFA, que demonstrou menor mobilização lipídica no mesmo grupo.

Abstract The prevalence of obesity is dramatically on the rise in the Western world. Obesity is associated with several chronic diseases, including diabetes and cardiovascular disease (CVD). Non-alcoholic fatty liver disease occurs when fat is ectopically stored in the liver. It is closely associated with serious metabolic abnormalities. Non-alcoholic fatty liver disease ranges from simple hepatic steatosis with no inflammation to hepatic steatosis with a necroinflammatory component, which may lead to cirrhosis and liver failure. Adiponectin is an adipokine that is solely secreted by adipocytes and has anti-inflammatory, antiatherogenic and insulin-sensitizing properties. Adipose tissue inflammation contributes to reduced plasma adiponectin levels in obesity leading to further metabolic complications. Adiponectin may be a mediator between obesity and fat accumulation in the liver and skeletal muscle. Fatty liver may play a role in the pathogenesis of CVD. Mortality data show that CVD as the cause of death accounts for almost half of all deaths in Finland. Traditional risk factors for CVD are age, gender, smoking, high low-density lipoprotein level, high blood pressure and diabetes. The aim of the thesis was to investigate the mediators of fat accumulation in the liver and skeletal muscle as well as the role of fatty liver in the future risk for CVD. If one considers the peptide hormones, then adiponectin turned out to be the strongest independent indicator of the brightness of the liver. In addition, an association between a low adiponectin concentration and large muscle fiber size was observed, and this was not dependent on the amount of total fatness. Furthermore, severe fatty liver increased the risk for cardiovascular events, predicted the risk for death from all causes and death from CVD in a long follow-up. Insulin sensitivity seemed to play a more dominant role in developing cardiovascular events. In conclusion, this study demonstrates that adiponectin may have an important effect on fat accumulation in the liver and skeletal muscle. Adiponectin could be a target when considering the treatment and prevention of ectopic fat accumulation. Fatty liver seems to play a significant role in developing cardiovascular event and mortality to CVD. / Tiivistelmä Lihavuus on kasvava ongelma länsimaissa. Lihavuudella on todettu olevan yhteyttä lukuisiin kroonisiin sairauksiin, kuten diabetekseen ja sydän- ja verisuonitautiin. Ei-alkoholiperäinen rasvamaksa aiheutuu rasvan kertymisestä maksaan. Tilan on todettu liittyvän läheisesti vaikeisiin aineenvaihdunnan häiriöihin. Ei-alkoholiperäinen rasvamaksa vaihtelee vakavuusasteeltaan poikkeavasta rasvan kertymisestä tulehdukseen, joka voi edelleen johtaa kirroosiin ja maksan toiminnan pettämiseen. Adiponektiini on pääasiassa rasvakudoksen erittämä hormoni, jolla on tulehdusta hillitseviä, ateroskleroosilta suojaavia ja insuliinia herkistäviä ominaisuuksia. Rasvakudoksen tulehdustila myötävaikuttaa alentuneeseen adiponektiinipitoisuuteen, joka voi johtaa vaikeutuneisiin aineenvaihdunnan häiriöihin. Adiponektiinin epäillään olevan välittäjäaine lihavuuden ja rasvamaksan ja lihaksensisäisen rasvan välillä. Rasvamaksan ja kardiovaskulaarisairauksien välillä saattaa olla yhteys. Sydän- ja verisuonisairaudet aiheuttavat lähes puolet kuolemista Suomessa. Perinteisiä kardiovaskulaaritaudin riskitekijöitä ovat ikä, sukupuoli, tupakointi, korkea LDL-kolesteroli, korkea verenpaine ja diabetes. Tutkimuksemme tavoitteena oli selvittää maksan ja lihaksen rasvan kertymiseen myötävaikuttavia tekijöitä sekä rasvamaksan vaikutusta riskiin sairastua sydän- ja verisuonisairauksiin. Tutkimuksessa havaittiin, että lihavuuteen liittyvistä hormoneista adiponektiini oli vahvin itsenäinen myötävaikuttaja rasvamaksan kehittymisessä. Plasman alentunut adiponektiinipitoisuus yhdistyi kasvaneeseen lihassolun kokoon riippumatta henkilöiden rasvakudoksen määrästä. Seurantatutkimuksen mukaan vaikeasti rasvoittunut maksa lisäsi riskiä sairastua kardiovaskulaaritautiin, ennusti yleistä kuolemanriskiä ja kuolemaa kardiovaskulaaritautiin. Insuliiniherkkyydellä näytti olevan merkittävä rooli sydän- ja verisuonitautitapahtumissa. Tutkimus osoittaa, että adiponektiinillä saattaa olla keskeinen rooli rasvan kertymisessä maksaan ja lihakseen. Adiponektiini voi olla keskeinen tutkimuskohde kehiteltäessä hoitomuotoja ja ehkäisymenetelmiä rasvakudoksen ulkopuolisen rasvan kertymiseen. Rasvamaksan rooli sairaalahoitoon tai kuolemaan johtavissa ateroskleroottisissa tapahtumissa on ilmeinen.

L’infection par le virus de l’hépatite C (HCV) est une cause majeure d’hépatite chronique, de cirrhose hépatique et ce carcinome hépatocellulaire dans le monde. La compréhension des relations entre le virus et sa cellule hôte, l’hépatocyte, est indispensable pour le développement de nouvelles stratégies thérapeutiques et d’un vaccin préventif. La particularité de ce virus est son lien étroit avec le métabolisme lipidique. Le virus circule dans le sang associé aux lipoprotéines, formant une lipo-viro-particule (LVP) infectieuse. L’apolipoprotéine E (apoE) est une protéine cellulaire faisant intégralement partie de la LVP. Elle joue un rôle majeur dans l’infection et à la production des particules virales. Durant ce travail de thèse, j’ai pu approfondir les connaissances sur le rôle d’apoE dans le cycle viral du HCV sous deux aspects très différents. D’une part, j’ai pu démontré que la forme libre d’apoE, non liée aux lipoprotéines, inhibe la réplication du HCV grâce à la régulation du métabolisme lipidique hépatique, en induisant un efflux de cholestérol ABCG1-dépendant. D’autre part, j’ai participé à une étude démontrant que l’apoE liée à la LVP contribue à l’échappement du virus au système immunitaire, en masquant les épitopes de la protéine virale E2 aux anticorps neutralisants. Ces études ont mis en évidence deux nouveaux rôles d’apoE dans la pathogenèse du HCV. / Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. Understanding virus-host interactions in hepatocytes will contribute towards the development of new therapeutic strategies and a protective vaccine. HCV life cycle and the lipid metabolism are inextricably intertwined. A particular feature of this virus is that, in the blood, HCV virions are associated with lipoproteins, forming an infectious lipoviroparticle (LVP). Apolipoprotein E (apoE) is a key component of LVPs that plays an essential role in HCV entry and virions production. My PhD project entailed a more detailed dissection of apoE’s role in the HCV life cycle. I demonstrated that lipid-free apoE inhibits HCV replication by regulating the hepatic lipid metabolism via an ABCG1-dependent cholesterol efflux. Furthermore, I contributed to a study demonstrating that LVP-associated apoE helps the virus escape host immunity by blocking access of neutralizing antibodies to the viral glycoprotein E2. The work presented highlights two new roles of apoE in HCV pathogenesis

La prévalence du syndrome métabolique (MetS) est en constante augmentation dans les pays industrialisés. La stéatose hépatique, caractérisée par une accumulation massive de lipides dans les hépatocytes est une des manifestations du MetS. Parmi les SNP (Single Nucleotide Polymorphism) associés au MetS et à la stéatose hépatique, le SNP I148M de l’adiponutrine/PNPLA3 (Patatin-like Phospholipase Domain-Containing) est décrit comme un nouveau marqueur de la stéatose et également de la sévérité des différentes atteintes hépatiques des NAFLD (Non-alcoholic Fatty Liver diseases). L’objectif de ma thèse a été de déterminer le rôle de l’adiponutrine dans le foie, d’une part, en étudiant sa régulation transcriptionnelle par les facteurs de transcription SREBP1c (Sterol Responive Element Binding Protein) et ChREBP (Carbohydrate Responsive Element Binding Protein) en réponse à l’insuline et au glucose et d’autre part, en étudiant l’impact de sa surexpression sur le métabolisme glucido-lipidique dans le foie de souris. L’adiponutrine est régulée de façon semblable aux enzymes lipogéniques et semble avoir un impact sur le métabolisme lipidique dans le foie. Comme l’adiponutrine, différents SNP des médiateurs de la voie Wnt/β-caténine sont également associés au syndrome métabolique et au diabète. La voie Wnt/β-caténine joue un rôle déterminant dans la zonation du lobule hépatique. Nous nous sommes intéressés à la régulation de cette voie par les conditions nutritionnelles et dans un contexte physiopathologique de stéatose et de résistance à l’insuline. Nos résultats montrent la régulation de cette voie par les hormones pancréatiques (insuline et glucagon) dans le foie favorisant ainsi l’orientation métabolique des hépatocytes en fonction des besoins. De plus, cette voie est dérégulée dans le foie d’animaux résistants à l’insuline, suggérant qu’elle pourrait avoir une fonction dans ce désordre métabolique. / The prevalence of metabolic syndrome (MetS) has increased in industrial countries. The hallmark of MetS in the liver is an excessive accumulation of triglyceride, which is called hepatic steatosis. Different SNP (Single Nucleotide Polymorphism) are associated with hepatic steatosis or MetS. One of them is found on adiponutrin/PNPLA3 (Patatin-like Phospholipase Domain-Containing) gene (SNP I148M) and is now considered as a new marker of hepatic steatosis and severity of NAFLD (Non-alcoholic Fatty Liver diseases). In order to understand the physiological role of adiponutrin in the liver, we studied its transcriptional regulation by SREBP1c (Sterol Responive Element Binding Protein) and ChREBP (Carbohydrate Responsive Element Binding Protein), mediators of insulin and glucose respectively. Moreover, by overexpressing adiponutrin in mice liver, we investigated its role in hepatic carbohydrate and lipid metabolism. We showed that adiponutrin is regulated as lipogenic genes and could have a role lipid metabolism. As for adiponutrin I148M, different SNP are found on substrats of Wnt/β-catenin pathway, a major pathway controlling acinus zonation. We examined the regulation of this pathway by nutritionnal status and in a pathophysiological context of insulin resistance and steatosis. We showed that Wnt/β-catenin pathway is regulated by pancreatic hormones (insulin and glucagon) in the liver in order to adapt hepatocyte phenotype to energetic needs. Moreover, this pathway is dysregulated in insulin resistant mice liver. These data may suggest a link between Wnt/β-catenin pathway deregulation and hepatic metabolic disorders.

L'huile de lin est considérée comme une des plus riches en acide linolénique (oméga- 3), avec certaines variétés de lin oléagineux produisant jusqu'à 65% d'acide a-linolénique. Cette huile utilisée depuis longtemps en industrie chimique pour la confection de peintures, vernis, agents tensioactifs, présente aussi des bénéfices pour la santé humaine avec un apport journalier d'environ 2 g.f 1 (diminution de la pression artérielle, prévention de thrombose...). Il parait donc raisonnable aujourd'hui d'augmenter la production de l'huile de lin riche en oméga-3 pour répondre à ces différents besoins. La sélection de cultivars producteurs d'huile de la qualité demandée devient impérative. Pour cela, des connaissances sur les mécanismes de synthèse et d'accumulation AG polyinsaturés (PUFA) dans les graines de lin sont à acquérir. Dans ce contexte, nous avons évalué le rôle des enzymes de type LPAAT dans l'accumulation des PUFA dans le but de sélectionner des isoformes compétitives dans leur accumulation au niveau des TAG. Les gènes Lpaat ont été isolés à partir des graines de lin âgées de 20 JAF, phase active de remplissage en huile. La fonctionnalité des protéines correspondantes a été testée par complémentation dans une souche JC201 d’E. coli déficiente en activité LPAAT. Une caractérisation de ces enzymes a été effectuée par des tests in vitro et in vivo. Les résultats ont montré la présence d'une isoforme, nommée LPAAT2A, dont l'activité LPAAT présente une spécificité et sélectivité élevées vis-à-vis des PUFA. Dans un 2ème temps, nous avons mesuré in vitro et in vivo le potentiel des enzymes LPAAT provenant de litchi et de lin vis-à-vis des AG de type cyclopropanique. Ces AG possèdent des propriétés physico-chimiques intéressantes pour l'industrie des lubrifiants et des cosmétiques et sont produits par une enzyme spécifique « la CFA synthase ». Nous avons généré des lignées d'Arabidopsis exprimant la CFA synthase d’E.coli puis co-exprimé le gène Lpaat de litchi. Une augmentation de la teneur en AGC (AG cycliques) dans les lignées transgéniques obtenues reflète la spécificité de la LPAAT de litchi vis-à-vis des AGC. Nous nous sommes aussi intéressés aux enzymes de types sPLA2a (phospholipase A2). L'isolement du gène correspondant a été effectué à partir des graines de lin pendant la phase active de remplissage en huile. La caractérisation de son activité a été effectuée par des tests in vitro et in vivo. Les résultats ont montré que l'expression du gène sP/a2a de lin dans les graines d' Arabidopsis provoque une augmentation du poids de la graine ainsi qu'une augmentation du contenu en acide linolénique dans les TAG. L'ensemble de ces résultats montre l'existence d'un système enzymatique endogène chez le lin efficace vis-à­vis des PUFA et qui semble stimuler le métabolisme lipidique une fois exprimé chez Arabidopsis. / Flaxseed oil contain high amount of oméga-3 and present different industrials applications and human health benefits. ln this work, we aimed to identify enzymes allowing the accumulation of high level of omega-3 in linseed plants. ln this context, we evaluated the role of Lysophophatidic acid acyltransferases (LPAAT) and Phospholipases A2 (PLA2) enzymes in the accumulation of omega-3 by in vitro and in vivo approaches. Results showed the presence of a LPAAT2A isoform into flax genome having a high specificity and selectivity toward omega-3. Expression of Lpaat2A and sPia2a gene in Arabidopsis seeds increase seed weight, oil production and omega-3 content (up to 10% and 11%) in transformant seeds respectively. These results showed the presence in linseed plant of an efficient enzymatic system toward omega-3 accumulation. Furthermore, we have evaluated in vitro and in vivo the potential of Litchi and flax LPAAT enzymes toward the production of cyclopropane fatty acids (CFA). These fatty acids are naturally produced by a specific enzyme «CFA synthase» with physico-chemical properties interesting to lubricant and cosmetics industries. ln this context, we have generated Arabidopsis lines expressing E. coli Cfa synthase with or not co-expression of litchi Lpaat gene. Result showed an increase of 25% in the content of CFA in transgenic line co-expressing Cfa synthase and litchi Lpaat comparing to the transgenic lines expressing the CFA synthase. This increase in the level of CFA in transgenic seeds reflects the specificity of Litchi LPAAT toward CFA.

Animals rely on lipids as a major fuel for endurance exercise because they pack more joules per gram than any other fuel. However, in contrast to mammals, information on how the mobilization of lipids from endogenous stores is managed to meet the needs of energy metabolism in swimming fish is sparse. Information on in vivo rates of lipid mobilization in swimming fish has been limited to relatively low exercise intensities and has only been investigated in a single species. Therefore, the goal of my thesis was to address this paucity of information by quantifying lipolytic rate in rainbow trout during graded exercise and fatty acid mobilization in Atlantic salmon during prolonged endurance exercise. In the first part of my work, I hypothesized that like mammals, rainbow trout stimulate lipolysis above resting levels to a peak with increasing work intensity, but subsequently lower its rate at high intensities when ATP production from carbohydrates becomes dominant. To test this hypothesis, I measured the rate of appearance of glycerol (Ra glycerol) in the blood (resulting from the breakdown of triacylglycerol (TAG)) of trout at rest (control) and during graded exercise from rest to Ucrit. Results showed that Ra glycerol in trout averaged 1.24 ± 0.10 µmol kg -1 min-1 and that this rate was unaffected by exercise of any intensity. These experiments revealed that rainbow trout do not modulate lipolysis during exercise. Furthermore, I calculated that baseline lipolytic rate was much higher in trout than in mammals and that this rate is in constant excess of the requirements of energy metabolism. My second investigation focused on measuring fatty acid mobilization in Atlantic salmon. To date, the majority of studies on energy metabolism in salmonids have used rainbow trout as the ubiquitous model for salmonids. I postulated that domesticated rainbow trout may be far less impressive athletes than their wild anadromous form and other salmonids. In this regard, I proposed that studying energy metabolism in Atlantic salmon (even those from aquaculture) may help to deepen our understanding of the physiology of true long-distance migrant fish. To study the effects of prolonged endurance exercise on the mobilization of fatty acids from endogenous stores in these fish, I monitored the rate of appearance of fatty acids (Ra NEFA calculated from Ra Palmitate) in the blood during 72 hours of sustained swimming. I found that contrary to what has been previously described in rainbow trout, Ra Palmitate (and by proxy, Ra NEFA) is reduced by approximately 64% (from 0.75 ± 0.12 µmol kg-1min-1 to 0.27 ± 0.06 µmol kg-1min-1 and from 19.3 ± 7.8 µmol kg-1min-1 to 6.9 ± 2.0 µmol kg-1min-1 for Ra Palmitate and Ra NEFA, respectively) during prolonged endurance exercise in Atlantic salmon. However, like in trout, even this reduced rate of fatty acid mobilization exceeds the requirements of energy metabolism at rest and during swimming. While further experiments will be necessary, I speculated that this reduction in Ra NEFA may be caused by a partial inhibition of lipolysis to reduce the energetic cost of TAG:FA cycling and optimize fuel budgets during prolonged endurance exercise. This thesis provides the first in vivo measurements of lipolysis during graded exercise in salmonids and the first in vivo measurements of fatty acid mobilization in Atlantic salmon. From the results mentioned above, I concluded that salmonids mobilize lipids in constant excess of the requirements for energy metabolism, possibly to allow for rapid reorganization of membrane phospholipids in response to changing environmental conditions. However, more anadromous and migratory phenotypes may rely on a tighter control of lipolysis to minimize the costs of substrate cycling and conserve energy on limited fuel stores.